| Objective: Cerebrovascular diseases have a serious harm to health, and its incidence is very high constantly. Most of the cerebral vascular diseases were ischemic encephalopathy .The aim of treatment to cerebrovascular diseases was mainly to restore the blood supply of ischemic brain tissue. However, this process would inevitably induce the ischemia/reperfusion (I/R) injury to brain tissue.Currently, the main aim of the researchers was searching for the methods lessening or preventing cerebral ischemia/reperfusion injury. Ischemic preconditioning was reported by Murry and his colleague in 1986, they found that transient ischemia could induce a protective effect to myocardium against injury induced by a severity ischemia. That was defined to ischemic preconditioning. The research about preconditioning was started after that. Until now, several methods of preconditioning such as limb ischemic preconditioning, hypoxic preconditioning and ischemic preconditioning were used in research. In the 1990, it was reported that transient hypoxia could induce a resistant to the injury induced by severity ischemia in the rats. Recently, the researchers began to work in the protective effect of hypoxic preconditioning to tissue with ischemia/reperfusion. But until now, the mechanism of hypoxic preconditioning has not been understood.In this study, the rats were treated by intermittent hypobaric hypoxia preconditioning(IHHP), and then 4-vessel occlusion (4VO) was used to establish global cerebral ischemia model. The effect of IHHP on hippocampus neuron of rat with global cerebral ischemia/reperfusion(I/R)was observed . The protective effect of HO-1 expression induced by IHHP on hippocampus neurons of rats with global cerebral ischemia/ reperfusion was investigated. The first part:.The protective effect of intermittent hypobaric hypoxia preconditioningon hippocampus neurons of rats with cerebral ischemia / reperfusionMethods: 30 male wistar (280-300g ) rats were randomly divided into five groups with 6 rats in each group.①normal group: none of any treatment②sham group: Bilateral vertebral arteries were occluded firstly,bilateral common carotid arteries were exposed after 48 hours only but were not occluded.③Intermittent hypobaric hypoxia preconditioning(IHHP) group: rats were put in hypopietic hypoxia (70.66-70.93kp) environment for 30min, once a day for 4 days.④global cerebral ischemia/reperfusion(I/R)group: Bilateral vertebral arteries were occluded firstly, bilateral common carotid arteries were exposed after 48 hours and were occluded for 8 min and then the blood was reperfused.⑤IHHP+ I/R group: Rats in this group were treated with IHHP for 4 times with 1 time each day, bilateral vertebral arteries were occluded in 5th day, bilateral common carotid arteries were exposed after 48 hours and were occluded for 8 min and then the blood was reperfused. All the rats in each group were sacrificed by decapitation on 7th day after reperfusion, hippocampus tissue were collected and stained with thionine to decide survival neuron density in CA1 region of hippocampus.The neuronal density (ND) of the hippocampus CA1 subfield was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus and clear nucleolus within 1 mm linear length of the CA1. The average of number of pyramidal neurons in 3 areas of the hippocampus CA1 subfield was calculated as ND.Result: The neuronal density(ND)of normal group is 142.67±13.30 (number/mm2). It was found by thionine staining that there was no significant neuronal damage in the CA1 hippocampus of Sham and IHHP groups and neuronal density(ND)were 121.33±12.04 (number/mm2) and135.00±13.25, there was no significant difference compared with the normal group . The hippocampus CA1 subfield exhibited obvious destruction in I/R group, the ND (39.30±9.90) was significantly lower than that in the Sham group (P<0.01), suggesting that cerebral ischemia for 8 min caused serious neuronal death in the hippocampus CA1 . Compared with I/R group, pyramidal neuronal arrange in hippocampus CA1 were obviously amelioration in the rats of IHHP+I/R, the ND (89.33±13.50) was obviously increased compared with the I/R group (P<0.01).The second part: The protective effect of HO-1 induced by IHHP on the hippocampus neurons of rats with global cerebral ischemia / reperfusion. Method: 90 male wistar (280-300g) rats were divided into 2 groups.In the first group,the HO-1 expression induced by IHHP was detected in the hippocampus CA1 of rats. In the second group, the protective effect of HO-1 on the hippocampus neurons of rats with global cerebral ischemia / reperfusion was investigated.2.1 In the first group, 30male wistar (280-300g) rats were divided into five subgroups with 6 each group.①normal group,②Sham group,③Intermittent hypobaric hypoxia preconditioning (IHHP) group,④global cerebral ischemia/reperfusion (I/R) group and⑤IHHP+ I/R group. The treatments of the rats were as same as that in the first part. All these rats were sacrificed by decapitation on 24 hours after reperfusion, hippocampus tissue were collected. The HO-1 expression was observed by immunohistochemistry method.2.2 In the second group, 60 male wistar (280-300g) rats were divided into five subgroups with 12 each group.①IHHP+ I/R group : Rats in this group were treated with IHHP for 4 times with 1 time each day, bilateral vertebral arteries were occluded in 5th day, bilateral common carotid arteries were exposed after 48 hours and were occluded for 8 min and then the blood was reperfused②IHHP+ I/R+DMSO group : Rats in this group were treated with IHHP for 4 times with 1 time each day, bilateral vertebral arteries were occluded in 5th day, 50μg DMSO was injected into peritoneal cavity and 30minutes later bilateral vertebral arteries were occluded and after 48 hours ,bilateral common carotid arteries were occluded for 8 min and then the blood was reperfused. 50μg, 100μg and 150μg of Znpp were injected into peritoneal cavity of the rats in the IHHP+ I/R+50μgZnpp group, IHHP+ I/R+100μgZnpp group and IHHP+ I/R+150μg Znpp group respectively.The other treatments were as same as the rats of IHHP+ I/R+DMSO group.Result:1 The effect of IHHP on the expression of HO-1The total area and average optical density (AOD) of normal group was.146.96±10.94 and 0.05±0.03(μm2); There was no significant difference compared with the rats in the Sham, they were 328.52±220.48 and 0.12±0.07.The total area and average optical density (AOD) of IHHP group were was higher compared with the normal group(P<0.05). The total area and AOD in I/R group exhibited obvious higher in the CA1 subfield, were significantly higher than that in the Sham group (P<0.05), suggesting that IHHP could induce the expression of HO-1 in the CA1 of hippocampus. The total area and average optical density (AOD)were 1655.00±289.00 and 0.61±0.49 of IHHP+I/R group, there were obviously higher compared with the I/R group(P<0.05).2 The protective effect of HO-1 on the hippocampus neurons of rats with global cerebral ischemia / reperfusion2.1 The effect of Znpp on the expression of HO-1 in hippocampus CA1 of rats with ischemia-reperfusion.The total area and average optical density (AOD) of IHHP+I/R group were.1655.00±289.00 and 0.61±0.49(μm2) respectively ; There was no significant difference compared with IHHP+I/R+DMSO group (1667.00±121.77and 0.72±0.33) (P>0.05).The total area and AOD of rats of IHHP+I/R+50μgZnpp group were 617.96±37.00 and 0.21±0.13 respectively, they were significantly decreased compared with the IHHP +I/R group (P<0.05).The total area and AOD in IHHP+I/R+100μgZnpp group were significantly decreased than that in the IHHP+I/R+50μgZnpp group (P<0.05), the total area and AOD were 204.99±121.51 and 0.07.±0.04 respectively .The total area and AOD were 161.11±68.25 and 0.06±0.03 of IHHP+I/R+150μgZnpp group, there were no obviously difference compared with the IHHP+I/R+100μgZnpp group (P>0.05).2.2 The effect of Znpp on neuronal density of hippocampus CA1 of rats with ischemia-reperfusion.The ND of IHHP+I/R+DMSO group was 86.00±16.34, compared with IHHP+I/R group (89.33±13.50), there was no significant difference (P>0.05). The hippocampus neuron of IHHP+I/R+50μgZnpp group exhibited obvious destruction in the CA1 subfield, the ND (63.33±9.26) was significantly lower than that in the IHHP+I/R group (P<0.05), suggesting that 50μgZnpp caused neuronal death in the CA1 of hippocampus. Compared with IHHP+I/R+50μgZnpp group, Neurons were further destructed in hippocampus CA1 of rats in the IHHP+I/R+100μgZnpp group and the IHHP+I/R+150μgZnpp group, the ND were 39.33±9.93 and 40.00±9.79 respectively (P<0.05), but there was no obviously difference between the two groups (P>0.05)Conclusions:1 IHHP could not induce obviously injury to hippocampus neurons of rats, but it could play a protective role to hippocampus neurons against ischemia/ reperfusion injury.2 IHHP could up-regulate the HO-1 expression in the hippocampus of normal rats and rats with global cerebral ischemia/reperfusion.3 HO-1 has a protective effect on the hippocampus neurons of rats with global cerebral ischemia/reperfusion, which may be one of the mechanisms of IHHP protective role to hippocampus neurons of rats with global cerebral ischemia /reperfusion.
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